Intracellular staining for cytokines and parasites, combined with two-color FACS analysis, were used to examine the frequencies of IL-12 and TNF-alpha producing macrophages in response to Leishmania infection and/or activation with IFN-gamma/LPS. Regardless of whether bone marrow-derived or inflammatory, macrophages from genetically resistant or susceptible mouse strains were used for infection, and regardless of whether the cells were infected in vitro or in vivo using metacyclic promastigotes or amastigotes, the effect of the parasite on cytokine responses by their host macrophages was identical: 1) neither IL-12 nor TNF-alpha were produced in response to infection itself; 2) virtually every infected cell lost its ability to produce IL-12 in response to IFN-gamma/LPS; and 3) the TNF-alpha response of infected cells was unimpaired. These data, generated using inflammatory macrophages, and low level, in vivo infection, reinforce the physiologic relevance of prior observations regarding the selective and potent inhibitory effect that Leishmania parasites have on IL-12 induction pathways in host macrophages. We suggest that selective impairment of IL-12 induction underlies the delayed onset of cell-mediated control mechanisms that is typical of even self-limiting forms of leishmanial disease. Studies have been initiated to evaluate the toxicity, immunogenicity and efficacy of a killed Leishmania vaccine made by B!obras in Brazil plus rhuIL-12 in monkeys (Macaca mulatta). Monkeys receiving two intradermal inoculations of aluym absorbed killed vaccine plus IL-12 were completely protected against homologous challenge with L. amazonensis. The protection was correlated with high levels of IFN-gamma in serum and in culture supernatants of PBLs stimulated with antigen in vitro.